Sepsis and sepsis syndromes are life-threatening conditions and are leading causes of death in intensive care units. Sepsis is most commonly caused by Gram-negative bacteria. Lipopolysaccharide (LPS) is an important component of the bacteria wall that contributes to the pathogenesis of sepsis. Endothelial cells (ECs) are a major target of LPS and the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha). Lipopolysaccharide, IL- 1beta, and TNFalpha all directly activate ECs and elicit an array of EC responses, including the upregulation of cytokines, chemokines, adhesion molecules, and procoagulant proteins. Most of these pro-inflammatory responses are mediated by the transcription factor NF-KappaB, which is also important for cell survival. Endothelial cell activation is thus thought to play a critical role in the development of both host defense and the systemic inflammatory response in sepsis. Increasing evidence also indicates that EC apoptosis contributes to the pathogenesis of experimental sepsis. Indeed, it has been suggested that a significant component of the beneficial effects of activated protein C, the only approved drug for the treatment of severe sepsis in man, may be due to its anti-apoptotic effects on ECs . The cellular receptor transducing the LPS signal has been identified as Toll-like receptor(TLR)4, a member of the larger family of TLR receptors. In this proposal we will examine further the molecular mechanisms of pro-inflammatory, pro-survival, and pro-apoptotic signaling by TLR4 in EC in vitro and determine the role of EC activation and apoptosis in vivo using transgenic mice in experimental sepsis induced by cecal ligation and puncture (CLP). The specific aims are: 1) To determine the role of phosphoinositide-3 kinase (PI3-K) in EC survival following LPS/TLR4 stimulation; 2) To determine the mechanisms of LPS/TLR4-lnduced pro-apoptotic signaling in ECs; 3) To identify new components of the TLR4-induced TRAF6-signaling complex in ECs; and 4) To determine the effect of inducible, selective blockade of EC NF-KappaB activation and apoptosis on sepsis induced by CLP.